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in southern Italy; this species is one of the most common potential vectors in Europe. ... X. fastidiosa in Italy using periwinkle as an indicator plant Saponari et al.

J. Appl. Entomol.

ORIGINAL CONTRIBUTION

Transmission of Xylella fastidiosa by naturally infected Philaenus spumarius (Hemiptera, Aphrophoridae) to different host plants D. Cornara1, V. Cavalieri2, C. Dongiovanni3, G. Altamura2, F. Palmisano3, D. Bosco4, F. Porcelli1, R. P. P. Almeida5 & M. Saponari2 1 2 3 4 5

DiSSPA-UNIBA Aldo Moro, Sez. Entomologia e Zoologia, Bari, Italy CNR Istituto per la Protezione Sostenibile delle Piante, Sede di Bari, Bari, Italy Centro di Ricerca, Sperimentazione e Formazione in Agricoltura ‘Basile-Caramia’, Locorotondo, Bari, Italy DISAFA – Entomologia, Universita degli Studi di Torino, Grugliasco, Torino, Italy ESPM, University of California Berkeley, Berkeley, CA, USA

Keywords spittlebugs, vector transmission, Xylella fastidiosa Correspondence Maria Saponari (corresponding author), CNR Istituto per la Protezione Sostenibile delle Piante, Sede di Bari, Via Amendola, 165/A 70126 Bari, Italy. E-mail: [email protected] Received: July 17, 2016; accepted: August 21, 2016. doi: 10.1111/jen.12365 First and second authors equally contributed to the work.

Abstract The recent establishment of Xylella fastidiosa subspecies pauca in the southern Italian region of Apulia threatens agricultural crops and the environment. Olive is an important and widespread ancient crop in Italy and, so far, the most impacted host. The meadow spittlebug Philaenus spumarius (Hemiptera, Aphrophoridae) has been identified as a vector of X. fastidiosa in southern Italy; this species is one of the most common potential vectors in Europe. To generate disease management strategies, data on X. fastidiosa transmission by P. spumarius are necessary. Therefore, we carried out transmission experiments by using field-collected spittlebugs in 2014 and 2015 (5 and 11 collection dates, respectively), and transferring groups of insects immediately on to recipient plants. Various host plant species were tested: olive, oleander, sweet orange, grapevine and the stone fruit rootstock GF677 (Prunus persica 9 Prunus amygdalus). Xylella fastidiosa was detected in all the host plants after insect plant access except for grapevine; infections to sweet orange and stone fruit were not systemic. In 2015, estimates of insect X. fastidiosa infectivity were obtained; the number of PCR-positive P. spumarius on each plant was positively correlated with the plant infection status. The proportion of P. spumarius infected with X. fastidiosa ranged from 25% to 71% during the entire survey period. The number of X. fastidiosa cells detected in P. spumarius heads ranged from 3.5 9 10 to 4.0 9 102 (CFU equivalents), which is lower than that reported for leafhopper vectors in the Americas. These data show that field-collected P. spumarius have high rates of X. fastidiosa infection and are competent vectors.

Introduction Pathogen introduction is often regarded as the main driver of emerging infectious diseases, although an arthropod-borne pathogen accidentally introduced in a new region requires a vector for establishment (Fereres 2015). The introduction of Xylella fastidiosa into Europe (Martelli et al. 2016) is an example of a

biological invasion that required endemic competent vectors for establishment (Almeida and Nunney 2015). In 2013, olive trees (Olea europaea) on the west coast of the Salento Peninsula (Apulia, Italy) showing leaf scorch and dieback, that is an unknown disease named ‘olive quick decline syndrome’ (OQDS), were found to be infected with X. fastidiosa subspecies pauca (Saponari et al. 2013). Xylella fastidiosa has been

© 2016 The Authors. Journal of Applied Entomology Published by Blackwell Verlag GmbH. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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detected in olive in the USA (Krugner et al. 2014; X. fastidiosa subsp. multiplex), Argentina (Haelterman et al. 2015; X. fastidiosa subsp. pauca) and more recently in Brazil (Coletta-Filho et al. 2016; X. fastidiosa subsp. pauca). There are limited data on X. fastidiosa infecting olives, but evidence indicates that pathogen genotype defines pathogenicity. While X. fastidiosa is associated with but does not cause disease in olives in the USA (Krugner et al. 2014), Koch’s postulates have been fulfilled in Italy (Saponari et al. 2016); pathogenicity data are not available from Brazil or Argentina. Only one genotype, based on multilocus sequence typing, has been recovered from olive trees and other hosts in Italy (Loconsole et al. 2016). The genotype is an identical match to X. fastidiosa subsp. pauca strain ST53, reported from Costa Rica in 2014 (Nunney et al. 2014; Giampetruzzi et al. 2015). In Italy, ST53 has been detected in cherry (Prunus avium L. 1755), myrtle-leaf milkwort (Polygala myrtifolia L.), coastal rosemary (Westringia fruticosa Willd.), acacia (Acacia saligna Labill.) and Spanish broom (Spartium junceum L.). To date, the host range of this genotype includes 22 plant species (Loconsole et al. 2016). It has been demonstrated that the meadow spittlebug Philaenus spumarius was a vector of X. fastidiosa in Italy using periwinkle as an indicator plant Saponari et al. (2014); it has also been demonstrated that P. spumarius transmits X. fastidiosa from infected to uninfected olive trees (Cornara et al. 2016a). Unfortunately, this vector species is ubiquitous and highly polyphagous; the nymphs can feed on huge varieties of herbaceous plants, while the adults disperse to an even higher number of plant species, including many trees and shrubs (Weaver and King, 1954; Delong and Severin 1950). In the contaminated area, olive is the predominant host in the cultivated areas, although other perennial hosts have been identified, mainly in non-cultivated areas, such as gardens and natural landscape. However, since both X. fastidiosa and P. spumarius have a wide host range (EFSA 2015), and plant community composition is important in the epidemiology of X. fastidiosa -associated diseases, we conducted studies in order to (i) evaluate the transmission of X. fastidiosa by P. spumarius to different host plants species and cultivars; and (ii) assess P. spumarius natural infectivity in olive orchards infected by X. fastidiosa. Material and Methods Plants

Plants used for the transmission experiments included different species and cultivars: (i) olive (Olea europaea) 2

cv. Coratina, cv. Ogliarola (two autochthonous olive cultivars widely grown in the Apulia region) and olive seedlings obtained from seeds of the cv. Cima di Mola; (ii) oleander (Nerium oleander); (iii) sweet orange (Citrus sinensis) cv. Madame vinous; (iv) grapevine (Vitis vinifera) cv. Cabernet Sauvignon; (v) stone fruit rootstock GF677 (hybrid Prunus persica 9 Prunus dulcis), commonly used for stone fruits breeding; and (vi) periwinkle (Catharanthus roseus). For the two olive cultivars and grapevines, experimental plants consisted of self-rooted cuttings; for the stone fruit rootstock GF677, they consisted of micropropagated plantlets, whereas seedlings were used for sweet orange cv. Madame Vinous and olive. All the plants were grown in a greenhouse located on the premises of the Campus of the University of Bari (in the pestfree area). Plants of 15–20 cm with young vegetating apexes were then transferred in a screenhouse in the infected area where the transmission tests were carried out. Transmission tests

We collected adult P. spumarius every 2 weeks from May to October, from infected olive orchards in Gallipoli (Apulia, Italy), by sweeping net and mouth aspirator. Insects were collected either from canopy or suckers of olive trees with severe symptoms of desiccation and dieback. Two distinct olive groves severely affected by OQDS (with 100% of symptomatic trees), one in 2014 and another one in 2015, were selected for insect collection; in both olive groves, the presence of X. fastidiosa in olive trees (ca. 200) was confirmed by real-time quantitative polymerase chain reaction (qPCR) (data not shown). Spittlebugs were temporarily stored in groups of five individuals in aerated plastic vials and brought to an insect-free transfer room. We caged five randomly selected spittlebugs on each recipient plant. Cages were made with fine mesh net sealed at the superior rim of the pot with two rubber bands. Transmission tests took place in an insect-proof screenhouse. A 4-day and 7-day inoculation access period (IAP) and 7-day IAP were used in 2014 and 2015, respectively. For each treatment (i.e. host plant), five replicates were performed; five plants of the same species/cultivar for each date with five insects per plant, except for periwinkle, for which two replicates per date were used. At the end of the IAP, insects were removed from the plants and stored in 75% ethanol. P. spumarius was identified according to Ossiannilsson (1981). Plants were treated with a systemic insecticide (Imidacloprid, Confidor 200 SL), stored in an insect-free screenhouse and tested by

© 2016 The Authors. Journal of Applied Entomology Published by Blackwell Verlag GmbH.

X. fastidiosa transmission by P. spumarius

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qPCR. In 2015, the presence and the population size of X. fastidiosa in P. spumarius were determined by qPCR in insects surviving the IAP. All the protocols are described below. Details of transmission assays for 2014 and 2015, including insect collection dates, plant species and number of tested plants, and insect and plant infection status after experiments, are available in Tables S1 and S2. Xylella fastidiosa detection in plants and insects

Plants were tested for the presence of X. fastidiosa by qPCR, using the assay developed by Harper et al. (2010). In 2014, all plants were sampled and tested 3, 6 and 14 months after the end of the last transmission tests (19 September 2014). Similarly, plants exposed to infective P. spumarius in 2015 were sampled and tested 3 and 6 months after the last transmission experiment (14 October 2015). Samples consisted of 4–6 leaves representative of the entire plant (collected from the bottom to the top of the main stem). Recipient plants that tested positive were resampled to assess the bacterial spread and the host colonization following the plant growth. Specifically, from each recipient qPCR-positive plant, leaves from the portion exposed to insect feeding and from the new growth were collected and tested separately (table 1). Spittlebugs were tested individually for the presence of X. fastidiosa. Briefly, the head of each insect was excised, the DNA extracted using a CTAB-based protocol (Loconsole et al. 2014) and SYBR-Greenbased qPCR assays performed using the primers targeting the conserved hypothetical HL protein (Francis et al. 2006); post-amplification melting curve analysis was carried out to assess the specificity of the amplification products. In 2015, at each date of collection, absolute quantification of the bacterial population harboured in the head of each P. spumarius was performed on five randomly selected qPCR-positive spittlebugs. Quantification cycle (Cq) value was plotted against a standard curve generated using serial 10-fold dilutions ranging from 10 to 107 CFU/ml of a bacterial suspension of Salentinian strain (CoDiRO) of X. fastidiosa. Statistical analysis

IBM SPSS Statistics 20 (SPSS 2012) was used for statistical analysis. To test for differences in transmission rate among species/cv used as recipient plants, and for differences related to the period when the transmission tests were carried out, data

Table 1 Detection of Xylella fastidiosa in leaf samples of the recipient plants. Samples were collected at the insect feeding site and on the new growth developed after the transmission experiment. Transmission experiments of 2014 and 2015

Species Olea europaea cv. Coratina Olea europaea cv. Ogliarola Olive seedling Prunus persica 9 Prunus dulcis (hybrid GF677) Nerium oleander Citrus sinensis cv. Madame Vinous Vitis vinifera cv Cabernet Sauvignon Catharanthus roseus

No. of total positive plants

No. of positive samples for the corresponding plant part* (a)

(b)

8

8

8

24

24

24

30 1

30 1

30 0

37 10

37 10

37 0

0

0

0

9

9

9

*Part (a) refers to the basal part of the plant (ca. 15 cm) including the leaves exposed to the insect feeding; part (b) refers to the plant parts above 15 cm including the new growth developed after the transmission experiment.

were analysed with the GENMOD procedure using the binomial distribution and the logit as link function in SAS (version 9.4, SAS Institute Inc., Cary, NC, USA). A likelihood ratio test was used to determine whether the host species or period when transmission test was performed affected (P < 0.05) transmission rate. The statistical significance (P < 0.05) of the likelihood ratio was determined by a chi-square test and least square means were used to determine significant differences (P < 0.05) among treatments (Agresti 2007). Hosts to which there was no detected transmission were excluded from statistical analysis. The 2014 and 2015 data sets were analysed independently. For olive seedlings, oleander and citrus, we assessed possible differences in transmission rate related to the different IAP (i.e. 4 and 7 days) through one-way ANOVA. Furthermore, for 2015, olive cv. Ogliarola and olive seedling, oleander and periwinkle tested plants, we evaluated the effect of the number of PCR-positive P. spumarius in each group (or plant) on the plant infectious status (0 = negative; 1 = positive) through binary logistic regression.

© 2016 The Authors. Journal of Applied Entomology Published by Blackwell Verlag GmbH.

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Results Transmission tests

In 2014, transmission rates differed significantly among host species (P < 0.05), although grapevines and the GF677 rootstocks were not included in the analysis as there was no detection of X. fastidiosa in those hosts (fig. 1). Transmission rate was highest for oleander (72.00  8.00), decreasing significantly (P = 0.0484) for olive seedling (44.00  13.27) and olive cv. Coratina (P = 0.0060) (32.00  14.97) with no significant differences between them (P = 0.3836), being lowest for sweet orange (16.00  16.00). Lastly, there was no difference in overall transmission rate related to the period when transmission tests were carried out in 2014 (P = 0.356), except for that performed in August that showed a significantly lower value than those sampled in April (P = 0.0026) or September (P = 0.0242). In 2015, treatments with X. fastidiosa infection were statistically different (P < 0.05). Transmission to GF677 rootstock, with only one plant infected in mid-June 2015, was significantly lower than to olive cv. Ogliarola (P = 0.0012), olive seedlings (P = 0.015), oleander (P < 0.0001) and periwinkle (P = 0.0010), but not differed for sweet orange (P = 0.1295). For those hosts, transmission rate was significantly higher for oleander (72.00  8.00) and periwinkle (50.00  11.79), decreasing significantly for olive cv. Ogliarola (43.64  10.02) and olive seedling (42.22  8.46) with no significant differences among them (P ≥ 0.05), being lowest (P < 0.0015) for sweet orange (12.00  5.33). There were no significant differences (P ≥ 0.05) in transmission rate related to the period when experiments were carried out, except for samples taken in mid-May and early June that showed a significantly lower transmission rate (P < 0.05). The increment in IAP from 4 days in 2014 to 7 days in 2015 had no effect on infection rate for olive seedlings (P = 0.8855), oleander (P = 0.7473) or sweet orange (P = 0.6320). Periodic diagnostic tests performed after both sets of transmission experiments showed that in both oleander and olives, bacterial spread occurred through the entire plants, including colonization of the new growth (table 1, Figure S1). For sweet orange, the bacterium was consistently detected only in the leaves and stems of the portion exposed to the infective insects, that is the 15 basal centimetre exposed to insect feeding. None of the samples (leaves and stem portions) collected from the new growths were positive for X. fastidiosa. Infection of the GF677 rootstock 4

occurred only in one out of 65 plants exposed during 2014 and 2015. Similarly to sweet orange, the bacterium was detected only in the portion exposed to the infective spittlebugs; additional tests failed in detecting the bacterium in the new growth. Infectivity of Philaenus spumarius used for the transmission tests

In 2015, a total of 1242 specimens used for transmission tests conducted from May to October were tested for the presence of X. fastidiosa; statistical difference among sampled population was observed (Student’s t = 8.02, d.f. = 10, P-value

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